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1.
《Channels (Austin, Tex.)》2013,7(5):346-357
Na+-coupled phosphate cotransporters of the SLC34 gene family catalyze the movement of inorganic phosphate (Pi) across epithelia by using the free energy of the downhill electrochemical Na+ gradient across the luminal membrane. Electrogenic (NaPi-IIa/b) and electroneutral (NaPi-IIc) isoforms prefer divalent Pi and show strict Na+:Pi stoichiometries of 3:1 and 2:1, respectively. For electrogenic cotransport, one charge is translocated per transport cycle. When NaPi-IIa or NaPi-IIb are expressed in Xenopus oocytes, application of the Pi transport inhibitor phosphonoformic acid (PFA) blocks a leak current that is not detectable in the electroneutral isoform. In this review, we present the experimental evidence that this transport-independent leak originates from a Na+-dependent uniport carrier mode intrinsic to NaPi-IIa/b isoforms. Our findings, based on the characteristics of the PFA-inhibitable leak measured from wild-type and mutant constructs, can be incorporated into an alternating access class model in which the leak and cotransport modes are mutually exclusive and share common kinetic partial reactions. 相似文献
2.
The type IIa Na+/Pi, cotransporter (NaPi-IIa) mediates electrogenic transport of three Na+ and one divalent Pi ion (and one net positive charge) across the cell membrane. Sequence comparison of electrogenic NaPi-IIa and IIb isoforms
with the electroneutral NaPi-IIc isoform pointed to the third transmembrane domain (TMD-3) as a possibly significant determinant
of substrate binding. To elucidate the role of TMD-3 in the topology and mechanism underlying NaPi-IIa function we subjected
it to cysteine scanning mutagenesis. The constructs were expressed in Xenopus oocytes and Pi transport kinetics were assayed by electrophysiology and radiotracer uptake. Cys substitution resulted in only marginally
altered kinetics of Pi transport in those mutants providing sufficient current for analysis. Only one site, at the extracellular end of TMD-3, appeared
to be accessible to methanethiosulfonate reagents. However, additional mutations carried out at D224 (replaced by E, G or
N) and N227 (replaced by D or Q) resulted in markedly altered voltage and substrate dependencies of the Pi-dependent currents. Replacing Asp-224 (highly conserved in electrogenic a and b isoforms) with Gly (the residue found in
the electroneutral c isoform) resulted in a mutant that mediated electroneutral Na+-dependent Pi transport. Since electrogenic NaPi-II transports 3 Na+/transport cycle, whereas electroneutral NaPi-IIc only transports 2, we speculate that this loss of electrogenicity might
result from the loss of one of the three Na+ binding sites in NaPi-IIa. 相似文献
3.
Nandhitha Subramanian Amanda J. Scopelitti Jane E. Carland Renae M. Ryan Megan L. O’Mara Robert J. Vandenberg 《PloS one》2016,11(6)
The Na+/Cl- dependent glycine transporters GlyT1 and GlyT2 regulate synaptic glycine concentrations. Glycine transport by GlyT2 is coupled to the co-transport of three Na+ ions, whereas transport by GlyT1 is coupled to the co-transport of only two Na+ ions. These differences in ion-flux coupling determine their respective concentrating capacities and have a direct bearing on their functional roles in synaptic transmission. The crystal structures of the closely related bacterial Na+-dependent leucine transporter, LeuTAa, and the Drosophila dopamine transporter, dDAT, have allowed prediction of two Na+ binding sites in GlyT2, but the physical location of the third Na+ site in GlyT2 is unknown. A bacterial betaine transporter, BetP, has also been crystallized and shows structural similarity to LeuTAa. Although betaine transport by BetP is coupled to the co-transport of two Na+ ions, the first Na+ site is not conserved between BetP and LeuTAa, the so called Na1'' site. We hypothesized that the third Na+ binding site (Na3 site) of GlyT2 corresponds to the BetP Na1'' binding site. To identify the Na3 binding site of GlyT2, we performed molecular dynamics (MD) simulations. Surprisingly, a Na+ placed at the location consistent with the Na1'' site of BetP spontaneously dissociated from its initial location and bound instead to a novel Na3 site. Using a combination of MD simulations of a comparative model of GlyT2 together with an analysis of the functional properties of wild type and mutant GlyTs we have identified an electrostatically favorable novel third Na+ binding site in GlyT2 formed by Trp263 and Met276 in TM3, Ala481 in TM6 and Glu648 in TM10. 相似文献
4.
Cristina Fenollar-Ferrer Monica Patti Thomas Knöpfel Andreas Werner Ian C. Forster Lucy R. Forrest 《Biophysical journal》2014
Phosphate plays essential biological roles and its plasma level in humans requires tight control to avoid bone loss (insufficiency) or vascular calcification (excess). Intestinal absorption and renal reabsorption of phosphate are mediated by members of the SLC34 family of sodium-coupled transporters (NaPi-IIa,b,c) whose membrane expression is regulated by various hormones, circulating proteins, and phosphate itself. Consequently, NaPi-II proteins are also potentially important pharmaceutical targets for controlling phosphate levels. Their crucial role in Pi homeostasis is underscored by pathologies resulting from naturally occurring SLC34 mutations and SLC34 knockout animals. SLC34 isoforms have been extensively studied with respect to transport mechanism and structure-function relationships; however, the three-dimensional structure is unknown. All SLC34 transporters share a duplicated motif comprising a glutamine followed by a stretch of threonine or serine residues, suggesting the presence of structural repeats as found in other transporter families. Nevertheless, standard bioinformatic approaches fail to clearly identify a suitable template for molecular modeling. Here, we used hydrophobicity profiles and hidden Markov models to define a structural repeat common to all SLC34 isoforms. Similar approaches identify a relationship with the core regions in a crystal structure of Vibrio cholerae Na+-dicarboxylate transporter VcINDY, from which we generated a homology model of human NaPi-IIa. The aforementioned SLC34 motifs in each repeat localize to the center of the model, and were predicted to form Na+ and Pi coordination sites. Functional relevance of key amino acids was confirmed by biochemical and electrophysiological analysis of expressed, mutated transporters. Moreover, the validity of the predicted architecture is corroborated by extensive published structure-function studies. The model provides key information for elucidating the transport mechanism and predicts candidate substrate binding sites. 相似文献
5.
Cristina Fenollar-Ferrer Monica Patti Thomas Kn?pfel Andreas Werner Ian?C. Forster Lucy?R. Forrest 《Biophysical journal》2014,106(6):1268-1279
Phosphate plays essential biological roles and its plasma level in humans requires tight control to avoid bone loss (insufficiency) or vascular calcification (excess). Intestinal absorption and renal reabsorption of phosphate are mediated by members of the SLC34 family of sodium-coupled transporters (NaPi-IIa,b,c) whose membrane expression is regulated by various hormones, circulating proteins, and phosphate itself. Consequently, NaPi-II proteins are also potentially important pharmaceutical targets for controlling phosphate levels. Their crucial role in Pi homeostasis is underscored by pathologies resulting from naturally occurring SLC34 mutations and SLC34 knockout animals. SLC34 isoforms have been extensively studied with respect to transport mechanism and structure-function relationships; however, the three-dimensional structure is unknown. All SLC34 transporters share a duplicated motif comprising a glutamine followed by a stretch of threonine or serine residues, suggesting the presence of structural repeats as found in other transporter families. Nevertheless, standard bioinformatic approaches fail to clearly identify a suitable template for molecular modeling. Here, we used hydrophobicity profiles and hidden Markov models to define a structural repeat common to all SLC34 isoforms. Similar approaches identify a relationship with the core regions in a crystal structure of Vibrio cholerae Na+-dicarboxylate transporter VcINDY, from which we generated a homology model of human NaPi-IIa. The aforementioned SLC34 motifs in each repeat localize to the center of the model, and were predicted to form Na+ and Pi coordination sites. Functional relevance of key amino acids was confirmed by biochemical and electrophysiological analysis of expressed, mutated transporters. Moreover, the validity of the predicted architecture is corroborated by extensive published structure-function studies. The model provides key information for elucidating the transport mechanism and predicts candidate substrate binding sites. 相似文献
6.
Bruce Felts Akula Bala Pramod Walter Sandtner Nathan Burbach Simon Bulling Harald H. Sitte L. Keith Henry 《The Journal of biological chemistry》2014,289(3):1825-1840
Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na+, Cl−, and K+ gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na+-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca2+ (but not other cations) to functionally replace Na+ for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca2+ and Na+ illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca2+ promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na+-binding sites. 相似文献
7.
Tatsiana Pakladok Zohreh Hosseinzadeh Aleksandra Lebedeva Ioana Alesutan Florian Lang 《The Journal of membrane biology》2014,247(2):137-145
B-RAF, a serine/threonine protein kinase, contributes to signaling of insulin-like growth factor IGF1. Effects of IGF1 include stimulation of proximal renal tubular phosphate transport, accomplished in large part by Na+-coupled phosphate cotransporter NaPi-IIa. The related Na+-coupled phosphate cotransporter NaPi-IIb accomplishes phosphate transport in intestine and tumor cells. The present study explored whether B-RAF influences protein abundance and/or activity of type II Na+-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb. cRNA encoding wild-type NaPi-IIa and wild-type NaPi-IIb was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type B-RAF, and electrogenic phosphate transport determined by dual-electrode voltage clamp. NaPi-IIa protein abundance in Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified by chemiluminescence. Moreover, in HEK293 cells, the effect of B-RAF inhibitor PLX-4720 on NaPi-IIa cell surface protein abundance was quantified utilizing biotinylation of cell surface proteins and western blotting. In NaPi-IIa-expressing Xenopus oocytes, but not in oocytes injected with water, addition of phosphate to extracellular bath generated a current (I P), which was significantly increased following coexpression of B-RAF. According to kinetic analysis, coexpression of B-RAF enhanced the maximal IP. Coexpression of B-RAF further enhanced NaPi-IIa protein abundance in the Xenopus oocyte cell membrane. Treatment of HEK293 cells for 24 h with PLX-4720 significantly decreased NaPi-IIa cell membrane protein abundance. Coexpression of B-RAF, further significantly increased IP in NaPi-IIb-expressing Xenopus oocytes. Again, B-RAF coexpression enhanced the maximal IP. In conclusion, B-RAF is a powerful stimulator of the renal and intestinal type II Na+-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb, respectively. 相似文献
8.
Lars Borre Thorvald F. Andreassen Lei Shi Harel Weinstein Ulrik Gether 《The Journal of biological chemistry》2014,289(37):25764-25773
The dopamine transporter (DAT) belongs to the family of neurotransmitter:sodium symporters and controls dopamine (DA) homeostasis by mediating Na+- and Cl−-dependent reuptake of DA. Here we used two-electrode voltage clamp measurements in Xenopus oocytes together with targeted mutagenesis to investigate the mechanistic relationship between DAT ion binding sites and transporter conductances. In Li+, DAT displayed a cocaine-sensitive cation leak current ∼10-fold larger than the substrate-induced current in Na+. Mutation of Na+ coordinating residues in the first (Na1) and second (Na2) binding sites suggested that the Li+ leak depends on Li+ interaction with Na2 rather than Na1. DA caused a marked inhibition of the Li+ leak, consistent with the ability of the substrate to interact with the Li+-occupied state of the transporter. The leak current in Li+ was also potently inhibited by low millimolar concentrations of Na+, which according to our mutational data conceivably depended on high affinity binding to Na1. The Li+ leak was further regulated by Cl− that most likely increases Li+ permeation by allosterically lowering Na2 affinity. Interestingly, mutational lowering of Na2 affinity by substituting Asp-420 with asparagine dramatically increased cation permeability in Na+ to a level higher than seen in Li+. In addition to reveal a functional link between the bound Cl− and the cation bound in the Na2 site, the data support a key role of Na2 in determining cation permeability of the transporter and thereby possibly in regulating the opening probability of the inner gate. 相似文献
9.
The Na+/K+-ATPase exports 3Na+ and imports 2K+ at the expense of the hydrolysis of 1 ATP. In the absence of K+, it carries on electroneutral, Na+-dependent transient charge movement (also known as “electroneutral Na+/Na+ exchange mode”) and produces a transient current containing faster and slower components in response to a sudden voltage
step. Components with different speeds represent sequential release of Na+ ions from three binding sites. The effect of holding potential on slow charge movement was studied in the presence of different
concentrations of ADPi, Nai+ and Nao+ with the intention of improving our understanding of Nai+ binding. However, the manipulation of [ADP]i and [Na+]i did not cause as pronounced changes as predicted in the magnitude of charge movement (Q
tot), which indicated that our experimental conditions were not able to backwardly drive reaction across the energy barrier to
Nai+ release/rebinding steps. On the contrary, lowering [Na+]o caused evident dependence of Q
tot on holding potential, with characteristics suggesting that pumps were escaping from E2P through the uncoupled Na+ efflux activity. 相似文献
10.
Intersubunit bridging by Na+ ions as a rationale for the unusual stability of the c-rings of Na+-translocating F1F0 ATP synthases
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The oligomeric c-rings of Na+-translocating F1F0 ATP synthases exhibit unusual stability, resisting even boiling in SDS. Here, we show that the molecular basis for this remarkable property is intersubunit crossbridging by Na+ or Li+ ions. The heat stability of c11 was dependent on the presence of Na+ or Li+ ions. For equal stability, 10 times higher Li+ than Na+ concentrations were required, reflecting the 10 times lower binding affinity for Li+ than for Na+. In a recent structural model of c11, the Na+ or Li+ binding ligands are located on neighboring c-subunits, which thus become crossbridged by the binding of either alkali ion with a concomitant increase in the stability of the ring. Site-directed mutagenesis strengthens the essential role of glutamate 65 in the crossbridging of the subunits and also corroborates the proposed stabilizing effect of an ion bridge including aspartate 2. 相似文献
11.
Steffen Hartung Nicolas Bucher Joseph B. Franklin Anna M. Wise Linda Y. Lim Han‐Yi Chen Johanna Nelson Weker Maria‐E. Michel‐Beyerle Michael F. Toney Madhavi Srinivasan 《Liver Transplantation》2016,6(9)
Sodium‐ion batteries may become an alternative to the widespread lithium‐ion technology due to cost and kinetic advantages provided that cyclability is improved. For this purpose, the interplay between electrochemical and structural processes is key and is demonstrated in this work for Na2.46V6O16 (NVO) and Li2.55V6O16 employing operando synchrotron X‐ray diffraction. When NVO is cycled between 4.0 and 1.6 V, Na‐ions reversibly occupy two crystallographic sites, which results in remarkable cyclability. Upon discharge to 1.0 V, however, Na‐ions occupy also interstitial sites, inducing irreversible structural change with some loss of crystallinity concomitant with a decrease in capacity. Capacity fading increases with the ionic radius of the alkali ions (K+ > Na+ > Li+), suggesting that smaller ions stabilize the structure. This correlation of structural variation and electrochemical performance suggests a route toward improving cycling stability of a sodium‐ion battery. Its essence is a minor Li+‐retention in the A2+xV6O16 structure. Even though the majority of Li‐ions are replaced by the abundant Na+, the residual Li‐ions (≈10%) are sufficient to stabilize the layered structure, diminishing the irreversible structural damage. These results pave the way for further exploitation of the role of small ions in lattice stabilization that increases cycling performance. 相似文献
12.
Acid-sensing ion channels (ASICs) are proton-gated cation-selective channels expressed in the peripheral and central nervous systems. The ion permeation pathway of ASIC1a is defined by residues 426–450 in the second transmembrane (TM2) segment. The gate, formed by the intersection of the TM2 segments, localizes near the extracellular boundary of the plasma membrane. We explored the contribution to ion permeation and selectivity of residues in the TM2 segment of ASIC1a. Studies of accessibility with positively charged methanethiosulfonate reagents suggest that the permeation pathway in the open state constricts below the gate, restricting the passage to large ions. Substitution of residues in the intracellular vestibule at positions 437, 438, 443, or 446 significantly increased the permeability to K+
versus Na+. ASIC1a shows a selectivity sequence for alkali metals of Na+>Li+>K+≫Rb+>Cs+. Alanine and cysteine substitutions at position 438 increased, to different extents, the relative permeability to Li+, K+, Rb+, and Cs+. For these mutants, ion permeation was not a function of the diameter of the nonhydrated ion, suggesting that Gly-438 encompasses an ion coordination site that is essential for ion selectivity. M437C and A443C mutants showed slightly increased permeability to K+, Rb+, and Cs+, suggesting that substitutions at these positions influence ion discrimination by altering molecular sieving. Our results indicate that ion selectivity is accomplished by the contribution of multiple sites in the pore of ASIC1a. 相似文献
13.
《Comparative biochemistry and physiology. B, Comparative biochemistry》1983,74(4):921-925
- 1.1. The behaviour of the Na efflux towards Li+ was studied using single barnacle muscle fibres as a preparation.
- 2.2. It is found that the Na efflux into Li+-ASW (artificial seawater) is reduced and that this effect is not fully reversed by returning back to Na+-ASW.
- 3.3. Preinjection of 100 mM-EGTA reduces the magnitude of the fall of the Na efflux into Li+-ASW.
- 4.4(a). The remaining Na efflux into Li+-ASW is further reduced by external application of 10−4 M-ouabain. (b) The remaining Na efflux in ouabain-poisoned fibres is reduced by replacing Nae by Li+. However, some fibres show a rise rather than a fall.
- 5.5. Fibres loaded with NaCl (by injection) show a prompt and sustained stimulation of the Na efflux when Nae is replaced by Li+. A similar but less pronounced response is often seen with ouabain-poisoned fibres.
- 6.6. Injection of LiCl (e.g. a 2 M-solution), causes a 20% fall in Na efflux. Subsequent replacement of Nae by Li+ fails to bring about a fall in the remaining efflux.
- 7.7. Itis concluded that the Na efflux in these fibres consists of a Na-Na exchange diffusion component which is not mediated by the Na-K pump and that its operation is interrupted by injecting Li+. The relative size of this component is about one-fifth and not one-half of the Na efflux.
14.
Ádám Fekete Laura Franklin Takeshi Ikemoto Balázs Rózsa Balázs Lendvai E. Sylvester Vizi Tibor Zelles 《Journal of neurochemistry》2009,111(3):745-756
Although the role of Na+ in several aspects of Ca2+ regulation has already been shown, the exact mechanism of intracellular Ca2+ concentration ([Ca2+]i) increase resulting from an enhancement in the persistent, non‐inactivating Na+ current (INa,P), a decisive factor in certain forms of epilepsy, has yet to be resolved. Persistent Na+ current, evoked by veratridine, induced bursts of action potentials and sustained membrane depolarization with monophasic intracellular Na+ concentration ([Na+]i) and biphasic [Ca2+]i increase in CA1 pyramidal cells in acute hippocampal slices. The Ca2+ response was tetrodotoxin‐ and extracellular Ca2+‐dependent and ionotropic glutamate receptor‐independent. The first phase of [Ca2+]i rise was the net result of Ca2+ influx through voltage‐gated Ca2+ channels and mitochondrial Ca2+ sequestration. The robust second phase in addition involved reverse operation of the Na+–Ca2+ exchanger and mitochondrial Ca2+ release. We excluded contribution of the endoplasmic reticulum. These results demonstrate a complex interaction between persistent, non‐inactivating Na+ current and [Ca2+]i regulation in CA1 pyramidal cells. The described cellular mechanisms are most likely part of the pathomechanism of certain forms of epilepsy that are associated with INa,P. Describing the magnitude, temporal pattern and sources of Ca2+ increase induced by INa,P may provide novel targets for antiepileptic drug therapy. 相似文献
15.
Carla P. Fonseca Luís L. Fonseca Liliana P. Montezinho Paula M. Alves Helena Santos M. Margarida C. A. Castro Carlos F. G. C. Geraldes 《European biophysics journal : EBJ》2013,42(7):503-519
Double quantum and triple quantum filtered 23Na nuclear magnetic resonance techniques were used to characterise in detail the isotropic and anisotropic binding and dynamics of intra- and extracellular Na+ in different cellular systems, in the absence and presence of Li+. The kinetics of Li+ influx by different cell types was evaluated. At steady state, astrocytes accumulated more Li+ than red blood cells (RBCs), while a higher intracellular Li+ concentration was found in chromaffin than in SH-SY5Y cells. Anisotropic and isotropic motions were detected for extracellular Na+ in all cellular systems studied. Isotropic intracellular Na+ motions were observed in all types of cells, while anisotropic Na+ motions in the intracellular compartment were only detected in RBCs. 23Na triple quantum signal efficiency for intracellular Na+ was SH-SY5Y > chromaffin > RBCs, while the reverse order was observed for the extracellular ions. 23Na double quantum signal efficiency for intracellular Na+ was non-zero only in RBCs, and for extracellular Na+ the order RBCs > chromaffin > SH-SY5Y cells was observed. Li+ loading generally decreased intracellular Na+ isotropic movements in the cells, except for astrocytes incubated with a low Li+ concentration and increased anisotropic intracellular Na+ movements in RBCs. Li+ effects on the extracellular signals were more complex, reflecting Li+/Na+ competition for isotropic and anisotropic binding sites at the extracellular surface of cell membranes and also at the surface of the gel used for cell immobilisation. These results are relevant and contribute to the interpretation of the in vivo pharmacokinetics and sites of Li+ action. 相似文献
16.
Zheyi Zou Nan Ma Aiping Wang Yunbing Ran Tao Song Yao Jiao Jinping Liu Hang Zhou Wei Shi Bing He Da Wang Yajie Li Maxim Avdeev Siqi Shi 《Liver Transplantation》2020,10(30)
Rhombohedral NaZr2(PO4)3 is the prototype of all the NASICON‐type materials. The ionic diffusion in these rhombohedral NASICON materials is highly influenced by the ionic migration channels and the bottlenecks in the channels which have been extensively studied. However, no consensus is reached as to which one is the preferential ionic migration channel. Moreover, the relationships between the Na+ distribution over the multiple available sites, concerted migration, and diffusion properties remain elusive. Using ab initio molecular dynamics simulations, here it is shown that the Na+ ions tend to migrate through the Na1–Na3–Na2–Na3–Na1 channels rather than through the Na2–Na3–Na3–Na2 channels. There are two types of concerted migration mechanisms: two Na+ ions located at the adjacent Na1 and Na2 sites can migrate either in the same direction or at an angle. Both mechanisms exhibit relatively low migration barriers owing to the potential energy conversion during the Na+ ions migration process. Redistribution of Na+ ions from the most stable Na1 sites to Na2 on increasing Na+ total content further facilitates the concerted migration and promotes the Na+ ion mobility. The work establishes a connection between the Na+ concentration in rhombohedral NASICON materials and their diffusion properties. 相似文献
17.
Olaf Sparre Andersen 《The Journal of general physiology》1998,111(1):1-18
The voltage dependence of the rat renal type II Na+/Pi cotransporter (NaPi-2) was investigated by expressing NaPi-2 in Xenopus laevis oocytes and applying the two-electrode voltage clamp. In the steady state, superfusion with inorganic phosphate (Pi) induced inward currents (Ip) in the presence of 96 mM Na+ over the potential range −140 ≤ V ≤ +40 mV. With Pi as the variable substrate, the apparent affinity constant (K
m
Pi) was strongly dependent on Na+, increasing sixfold for a twofold reduction in external Na+. K
m
Pi increased with depolarizing voltage and was more sensitive to voltage at reduced Na+. The Hill coefficient was close to unity and the predicted maximum Ip (Ipmax) was 40% smaller at 50 mM Na+. With Na+ as the variable substrate, K
m
Na was weakly dependent on both Pi and voltage, the Hill coefficient was close to 3 and Ipmax was independent of Pi at −50 mV. The competitive inhibitor phosphonoformic acid suppressed the steady state holding current in a Na+-dependent manner, indicating the existence of uncoupled Na+ slippage. Voltage steps induced pre–steady state relaxations typical for Na+-coupled cotransporters. NaPi-2-dependent relaxations were quantitated by a single, voltage-dependent exponential. At 96 mM Na+, a Boltzmann function was fit to the steady state charge distribution (Q-V) to give a midpoint voltage (V0.5) in the range −20 to −50 mV and an apparent valency of ∼0.5 e−. V0.5 became more negative as Na+ was reduced. Pi suppressed relaxations in a dose-dependent manner, but had little effect on their voltage dependence. Reducing external pH shifted V0.5 to depolarizing potentials and suppressed relaxations in the absence of Na+, suggesting that protons interact with the unloaded carrier. These findings were incorporated into an ordered kinetic model whereby Na+ is the first and last substrate to bind, and the observed voltage dependence arises from the unloaded carrier and first Na+ binding step. 相似文献
18.
Deciphering PiT transport kinetics and substrate specificity using electrophysiology and flux measurements 总被引:2,自引:0,他引:2
Ravera S Virkki LV Murer H Forster IC 《American journal of physiology. Cell physiology》2007,293(2):C606-C620
Members of the SLC20 family or type III Na+-coupled Pi cotransporters (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform a housekeeping function for intracellular Pi homeostasis. Previous studies have shown that PiT-1 and PiT-2 mediate electrogenic Pi cotransport when expressed in Xenopus oocytes, but only limited kinetic characterizations were made. To address this shortcoming, we performed a detailed analysis of SLC20 transport function. Three SLC20 clones (Xenopus PiT-1, human PiT-1, and human PiT-2) were expressed in Xenopus oocytes. Each clone gave robust Na+-dependent 32Pi uptake, but only Xenopus PiT-1 showed sufficient activity for complete kinetic characterization by using two-electrode voltage clamp and radionuclide uptake. Transport activity was also documented with Li+ substituted for Na+. The dependence of the Pi-induced current on Pi concentration was Michaelian, and the dependence on Na+ concentration indicated weak cooperativity. The dependence on external pH was unique: the apparent Pi affinity constant showed a minimum in the pH range 6.2–6.8 of 0.05 mM and increased to 0.2 mM at pH 5.0 and pH 8.0. Xenopus PiT-1 stoichiometry was determined by dual 22Na-32Pi uptake and suggested a 2:1 Na+:Pi stoichiometry. A correlation of 32Pi uptake and net charge movement indicated one charge translocation per Pi. Changes in oocyte surface pH were consistent with transport of monovalent Pi. On the basis of the kinetics of substrate interdependence, we propose an ordered binding scheme of Na+:H2PO4–:Na+. Significantly, in contrast to type II Na+-Pi cotransporters, the transport inhibitor phosphonoformic acid did not inhibit PiT-1 or PiT-2 activity. Na+-Pi cotransport; two-electrode voltage clamp; surface pH electrode; SLC20; retroviral receptor 相似文献
19.
Zhijian Huang 《Biophysical journal》2010,99(5):1416-1425
The transport cycle in the glutamate transporter (GlT) is catalyzed by the cotransport of three Na+ ions. However, the positions of only two of these ions (Na1 and Na2 sites) along with the substrate have been captured in the crystal structures reported for both the outward-facing and the inward-facing states of Gltph. Characterizing the third ion binding site (Na3) is necessary for structure-function studies attempting to investigate the mechanism of transport in GlTs at an atomic level, particularly for the determination of the sequence of the binding events during the transport cycle. In this study, we report a series of molecular dynamics simulations performed on various bound states of Gltph (the apo state, as well as in the presence of Na+, the substrate, or both), which have been used to identify a putative Na3 site. The calculated trajectories have been used to determine the water accessibility of potential ion-binding residues in the protein, as a prerequisite for their ion binding. Combined with conformational analysis of the key regions in the protein in different bound states and several additional independent simulations in which a Na+ ion was randomly introduced to the interior of the transporter, we have been able to characterize a putative Na3 site and propose a plausible binding sequence for the substrate and the three Na+ ions to the transporter during the extracellular half of the transport cycle. The proposed Na3 site is formed by a set of highly conserved residues, namely, Asp312, Thr92, and Asn310, along with a water molecule. Simulation of a fully bound state, including the substrate and the three Na+ ions, reveals a stable structure—showing closer agreement to the crystal structure when compared to previous models lacking an ion in the putative Na3 site. The proposed sequence of binding events is in agreement with recent experimental models suggesting that two Na+ ions bind before the substrate, and one after that. Our results, however, provide additional information about the sites involved in these binding events. 相似文献
20.
Aurelijus Zimkus Audrius Misiūnas Larisa Chaustova 《Central European Journal of Biology》2013,8(8):724-729
The effect of Li+ ions as a transformation inducing agent on the yeast cell wall has been studied. Two Saccharomyces cerevisiae strains, p63-DC5 with a native cell wall, and strain XCY42-30D(mnn1) which contains structural changes in the mannan-protein complex, were used. Fourier transform infrared (FT-IR) spectroscopy has been used for the characterization of the yeast strains and for determination of the effect of lithium cations on the cell wall. A comparison of the carbohydrate absorption band positions in the 970–1185 cm?1 range, of Na+ and Li+ treated yeast cells has been estimated. Absorption band positions of the cell wall carbohydrates of p63-DC5 were not influenced by the studied ions. On the contrary, the treatment of XCY42-30D(mnn1) cells with Li+ ions shifted glucan band positions, implying that the cell wall structure of strain XCY42-30D(mnn1) is more sensitive to Li+ ion treatment. 相似文献